Methods, systems and kits for in-pen assays
Methods, systems and kits are described herein for detecting the results of an assay. In particular, the methods, systems and devices of the present disclosure rely on a difference between the diffusion rates of a reporter molecule and an analyte of interest in order to quantify an amount of analyte in a microfluidic device. The analyte may be a secreted product of a biological micro-object.
1 . A method for determining a quantity of analyte secreted by a biological micro-object or a population of biological micro-objects generated therefrom, comprising:
(i) receiving imaging data of a microfluidic device, wherein
a. the microfluidic device includes a flow region comprising a microfluidic channel, and a plurality of sequestration pens, each comprising an isolation region and a connection region fluidically connecting the isolation region to the microfluidic channel at a single opening,
b. the imaging data includes an analyte assay image and one or both of a background noise image and a signal reference image,
c. the biological micro-object or the population of biological micro-objects generated therefrom secretes the analyte into the first fluidic medium within the sequestration pen, and
d. the analyte assay image comprises an image taken at a time point when the sequestration pen comprises a first fluidic medium, and a second fluidic medium has been introduced into the flow region for a first period of time, wherein the second fluidic medium comprises a plurality of reporter molecules, wherein each reporter molecule comprises: a binding component configured to bind the analyte, and a detectable label, thus allowing a portion of the plurality of reporter molecules to diffuse into each sequestration pen of the plurality and bind to at least a portion of the analyte secreted therein;
(ii) defining an area of interest for each sequestration pen, wherein the area of interest:
a. includes an image area within the microfluidic device, and at least a portion of the sequestration pen, and
b. extends along an axis of diffusion defined by the sequestration pen and the flow region; and
(iii) determining scores that are indicative of the quantity of analyte in each sequestration pen by analyzing at least a portion of the image area of the area of interest that extends along the axis of diffusion for each sequestration pen, wherein the score is correlated with a spatial distribution of the reporter molecule along the axis of diffusion.
2 . The method of claim 1 , wherein the area of interest is configured to capture a signal comprising a spatial distribution of fluorescence associated with diffusion along the axis of diffusion.
3 . The method of claim 1 , wherein the analyte binds to the reporter molecule forming a reporter molecule: secreted analyte (RMSA) complex.
4 . The method of claim 3 , wherein determining scores indicative of the quantity of analyte in each sequestration pen further comprises detecting the RMSA complexes located within the area of interest.
5 . The method of claim 4 , wherein detecting the RMSA complexes comprises measuring an intensity of a detectable signal coming from the area of interest, wherein at least some of the detectable signal emanates from the detectable label of reporter molecules located within the area of interest.
6 . The method of claim 5 , wherein detecting the RMSA complexes located within the area of interest further comprises:
measuring an intensity of a background signal from the background noise image within the area of interest; and
determining a background-subtracted signal intensity by subtracting the intensity of the background signal from the measured intensity of the detectable signal.
7 . The method of claim 6 , wherein measuring the intensity of the background signal within the area of interest is performed at a time prior to introducing the biological micro-object into the sequestration pen.
8 . The method of claim 6 , wherein the measured intensity of the detectable signal is normalized for a number of cells observed within the sequestration pen.
9 . The method of claim 1 , further comprising:
normalizing at least the image area of the area of interest for each of the sequestration pens in the analyte assay image by subtracting out background noise captured in the background noise image; and/or normalizing at least the image area of the area of interest for each of the sequestration pens in the analyte assay image by accounting for image acquisition distortions captured in the signal reference image,
wherein determining scores that are indicative of the quantity of analyte in each sequestration pen further comprises analyzing at least a portion of the normalized image area of the area of interest for each sequestration pen.
10 . The method of claim 9 , wherein determining scores that are indicative of the quantity of analyte in each sequestration pen further comprises applying a linear regression analysis to light emission data over at least a portion of the normalized image area of the area of interest of each sequestration pen.
11 . The method of claim 9 , wherein determining scores that are indicative of the quantity of analyte in each sequestration pen further comprises integrating light emission data over at least a portion of the normalized image area of the area of interest of each sequestration pen.
12 . The method of claim 1 , wherein the analyte comprises a protein.
13 . The method of claim 1 , wherein the connection region comprises a proximal opening to the microfluidic channel having a width W con of about 20 microns to about 100 microns and a distal opening to the isolation region, and wherein a length L con of the connection region from the proximal opening to the distal opening is at least 1.0 times the width W con of the proximal opening of the connection.
14 . A non-transitory computer-readable medium comprising executable instructions that, when executed causes the one or more processors of a computer to perform an image processing method for determining a quantity of analyte produced by a biological micro-object or population of biological micro-objects generated therefrom, the method comprising:
(i) receiving imaging data of a microfluidic device, wherein
a. the microfluidic device includes a flow region comprising a microfluidic channel, and a plurality of sequestration pens, each comprising an isolation region and a connection region fluidically connecting the isolation region to the microfluidic channel at a single opening,
b. the imaging data includes an analyte assay image and one or both of a background noise image and a signal reference image,
c. the biological micro-object or the population of biological micro-objects generated therefrom secretes the analyte into the first fluidic medium within the sequestration pen, and
d. the analyte assay image comprises an image taken at a time point when the sequestration pen comprises a first fluidic medium and a second fluidic medium has been introduced into the flow region for a first period of time, wherein the second fluidic medium comprises a plurality of reporter molecules, wherein each reporter molecule comprises: a binding component configured to bind the analyte, and a detectable label, thus allowing a portion of the plurality of reporter molecules to diffuse into each sequestration pen of the plurality and bind to at least a portion of the analyte secreted therein;
(ii) defining an area of interest for each sequestration pen, wherein the area of interest:
a. includes an image area within the microfluidic device, and at least a portion of the sequestration pen, and
b. extends along an axis of diffusion defined by the sequestration pen and the flow region; and
(iii) determining scores that are indicative of the quantity of analyte in each sequestration pen by analyzing at least a portion of the image area of the area of interest that extends along the axis of diffusion for each sequestration pen, wherein the score is correlated with a spatial distribution of the reporter molecule along the axis of diffusion.
15 . The non-transitory computer-readable medium of claim 14 , wherein the executable instructions further comprise instructions for:
normalizing at least the image area of the area of interest for each of the sequestration pens in the analyte assay image by subtracting out background noise captured in the background noise image; and/or normalizing at least the image area of the area of interest for each of the sequestration pens in the analyte assay image by accounting for image acquisition distortions captured in the signal reference image,
wherein determining scores that are indicative of the quantity of analyte in each sequestration pen further comprises analyzing at least a portion of the normalized image area of the area of interest for each sequestration pen.
16 . The non-transitory computer-readable medium of claim 15 , wherein the executable instructions further comprise determining scores that are indicative of the quantity of analyte in each sequestration pen by applying a linear regression analysis to the light emission data from at least a portion of the normalized image area of the area of interest of each sequestration pen.
17 . The non-transitory computer-readable medium of claim 15 , wherein the executable instructions further comprise determining scores that are indicative of the quantity of analyte in each sequestration pen by integrating light emission data over at least a portion of the normalized image area of the area of interest of each sequestration pen.